It is widely known that many chemotherapeutic regimens fail because the side-effects of the drugs used limit the dose that can be administered. This is particularly true of solid tumors. The clinically tolerated doses are often insufficient to kill all of the cells, thereby enriching the tumor population for drug resistant mutants. Among the surviving tumor cells in below-effective treatment regimens are mutant cells that arise spontaneously within the tumor cell population, and are resistant to the treatment drug. Each subsequent round of chemotherapy enriches the population for the resistant cells, which grow and continue to mutate, some to even higher levels of resistance. There is an established linear-log relationship between dose and tumor kill. The higher the dose of the drug, the greater the chance of eradicating the tumor. While methods have been developed to selectively target and kill tumor cells, many of the targeting methods either reduce the effectiveness of the drug, or call for a complex series of reactions to prepare a drug.
In the consideration of solid tumors, it should be recognized that local effective dosage, and systemic dosage, need not be the same. Thus, the only effective portion of the chemotherapeutic agent administered is that which reaches the tumor cell. Many chemotherapeutic agents are administered systemically, however, and only a limited portion (the local dosage) of the dosage administered actually reaches the cell. Thus, dose limitations frequently result in only a fraction of the permitted dosage actually reaching the cell.
The mechanism of inherent and acquired resistance of tumors to many forms of treatment involves glutathione. Elevated glutathione levels in tumors have been shown to contribute to resistance to chemotherapy and radiotherapy and prevent the initiation of the apoptotic cascade in tumor cells. The enzyme gamma-glutamyl transpeptidase (GGT, EC 2.3.2.2, also known as gamma-glutamyl transferase), which is localized to the cell surface, cleaves the γ-glutamyl bond of extracellular glutathione, releasing glutamic acid and cysteinyl-glycine, thus enabling the cell to use extracellular glutathione as a source of cysteine for increased synthesis of intracellular glutathione. GGT is induced in many human tumors, enhancing their resistance to chemotherapy. Inhibiting GGT prior to, during or after chemotherapy or radiation would sensitize GGT-positive tumors to treatment. Inhibiting GGT for as little as 2 hours lowers the intracellular cysteine concentration in GGT-positive tumors. However, all known glutamine analogs that inhibit GGT are too toxic for clinical use in humans at concentrations needed to inhibit GGT activity. Thus, identification of GGT inhibitors which could be used clinically has been a highly desired, yet unmet, need, until the present disclosure.
GGT is a cell surface enzyme that catalyzes the cleavage of the γ-glutamyl bond of glutathione (GSH), GSH-S conjugates, and leukotriene C4 (1, 23, 46). In humans, the expression of GGT is restricted predominantly to the apical surface of ducts and glands where fluids leave the body (2). The highest concentration of GGT is on the apical surface of the proximal tubule cells in the kidney where it prevents excretion of GSH into the urine by cleaving GSH present in the glomerular filtrate (2-3).
Catabolism of GSH by GGT affects intracellular redox levels and cysteine homeostasis [3, 47]. GGT cleavage of GSH S-conjugates alters drug toxicity and inflammation [48-50]. Overexpression of GGT has been implicated in pulmonary disease, cardiovascular disease and cancer [6, 8, 51]. Therefore, development of potent inhibitors of human GGT (hGGT) for clinical use would have broad therapeutic impact.
Aberrant expression and localization of GGT is observed in many disease states including cancer (4). Inhibiting GGT would sensitize tumors to chemotherapy and may be therapeutic in other diseases (5-8). However, GGT inhibitors that have been evaluated clinically are glutamine analogs and are neurotoxic (9-13). The inventors have previously reported the discovery of a novel inhibitor of GGT, referred to herein as OU749 (also referred to herein as Compound 1—see FIG. 6A below), which is not a glutamine analog (14). Inhibitors of hGGT that are considerably less toxic than the glutamine analogs are highly desirable.
GGT can catalyze hydrolysis reaction or a transpeptidation reaction. The first steps in both reactions are the cleavage of the γ-glutamyl bond of the substrate in the enzyme-substrate complex (ES) and the formation of a transient enzyme-glutamyl substrate complex (F-form of the enzyme). As the substrate is cleaved, the γ-glutamyl group forms a transient acyl bond with the enzyme, and the remainder of the substrate is released (15-17). In human GGT, the acyl bond forms between the γ-carbon of the γ-glutamyl substrate and the hydroxyl (beta-oxygen) on the side chain of Thr-381 (18). In the hydrolysis reaction, water hydrolyzes the acyl bond between the γ-glutamyl group and the nucleophilic residue releasing both glutamate and the enzyme (19-20). In the transpeptidation reaction, the γ-glutamyl group is transferred to the amine of an acceptor, thereby forming a new γ-glutamyl compound (21). The transpeptidation reaction occurs by a modified ping-pong mechanism (16, 22). The pH and amino acid concentrations in extracellular fluids where GGT is localized favor the hydrolysis reaction, and previous studies have indicated that the hydrolysis reaction is the predominant reaction catalyzed by GGT in vivo (22-23).
Expression of GGT on the surface of the cell initiates the cleavage of extracellular GSH thereby releasing cysteine and providing an additional source of cysteine for increased intracellular GSH synthesis (5, 37). GSH and free cysteine within the cell are potent reducing agents that protect the cell against oxidative stress and detoxify electrophilic metabolites. Conjugation of many chemotherapy drugs to GSH result in their inactivation, and the conjugates are exported from the cell (38). GSH has been shown to be increased in various cancers including breast (39), lung (40), bone marrow (41), ovarian (42), and head and neck laryngeal (43). Increased intracellular GSH has also been shown to contribute to the inhibition of apoptosis by inducing Bcl-2 and inducing resistance to antihormonal therapy (44-45). Studies have shown that, in mice, GGT-positive tumors are more resistant to treatment with cisplatin than GGT-negative tumors (6). In Phase I clinical trials, acivicin was found to be neurotoxic and cannot be used clinically as an inhibitor of GGT (10-13). Therefore, less toxic inhibitors of GGT are highly desired.
The most commonly used assay for GGT activity monitors the transpeptidation reaction with the synthetic compound L-gamma-glutamyl para-nitroanilide (L-GpNA) as the donor substrate and glycylglycine (GlyGly) as the acceptor. L-GpNA can also serve as a weak acceptor and therefore cannot be used in the absence of GlyGly to measure the hydrolysis reaction. D-gamma-glutamyl para-nitroanalide (D-GpNA), a stereoisomer of L-GpNA, cannot serve as an acceptor and is therefore used as a substrate to measure the hydrolysis reaction [18, 21]. However, the D-isomer of glutamate is not a physiological compound and there have been concerns as to the relevance of D-GpNA as a substrate. OU749 and certain structural analogs thereof are the only known uncompetitive inhibitors of the GGT transpeptidation reaction and the hydrolysis of D-GpNA [14, 53]. Uncompetitive inhibitors bind the gamma-glutamyl intermediate (F-form) of the enzyme. In the transpeptidation reaction, the inhibitors exhibited competitive inhibition with the dipeptide acceptor, GlyGly, indicating that they have a shared or overlapping binding site with GlyGly on the enzyme [14]. The inventors have developed a novel assay (L-Glutamate Release Assay) that monitors GGT hydrolysis of GSH and other physiological substrates [46].
OU749 (Compound 1) was previously identified by high-throughput screening of small molecules as inhibitors of the transpeptidation reaction [14]. Results are provided herein which demonstrate the potency and mechanism by which OU749 and other benzylthiadiazol sulfonamides inhibit GGT activity as measured by the transpeptidation reaction with the synthetic substrate, L-GpNA, the hydrolysis reaction with the synthetic substrate, D-GpNA, and the physiological hydrolysis reaction with GSH as the substrate.
Physicians generally prescribe three main treatments for cancer: surgery, radiation therapy, chemotherapy, or a combination of these. Surgery is generally advisable when physicians can safely remove the cancer from the body. In situations where the cancerous cells have spread, surgeons sometimes must remove large areas of healthy tissue along with the tumor to insure that no malignancy remains. In these cases, physicians may remove lymph nodes from the tumor area because cancer can spread through nodes. However, unfortunately many cancers are discovered too late for surgical cure. In many cases, the patient does not experience symptoms until the cancer has progressed to a malignant stage. Radiation therapy is used to destroy cancer cells. However, radiation can both cause and destroy cancer and can cause damage to surrounding tissues. Side effects of radiation therapy include radiation sickness, which are nausea and skin redness in the tumor area. Reducing the negative side effects of radiation treatment is therefore highly desirable.
Drugs used in chemotherapy take advantage of cancer cells' rapid growth and consumption of large amounts of nutrients. Side effects of chemotherapy can include nausea and temporary full or partial hair loss. Antimetabolites, one group of these drugs, work by mimicking the nutrients the body's cells consume. Physicians inject these drugs into the bloodstream, where they travel throughout the body, consumed by every cell. Rapidly growing cancerous cells consume much more of the poisonous drugs than do normal cells. As a result, the drugs destroy cancerous cells faster than normal cells. Another group of chemotherapy drugs interferes with the duplication of DNA (cells reproduce by duplicating their genetic code, or DNA), so cells cannot reproduce. Chemotherapy can also be directed against mutated proteins in the tumor cells, overexpressed proteins or other properties of the tumor cell. However, chemotherapy drugs may act on both the cancerous cells and the healthy cells. A physician's challenge is to administer the drugs so that only the cancer cells, and not the healthy cells, are killed. Side effects such as those described above prevent the long term or recurrent use of these drugs. Furthermore, there are an increasing number of effective drugs that can no longer be used due to resistance by the causative agent. It is thus highly desirable to reduce the side effects of therapeutics while maintaining the cancer-reducing qualities thereof, thus enabling: (a) longer term usage of the therapeutics, (b) usage of higher dosages of the therapeutics, or (c) enhancement of the therapeutics' effects so that lower dosages thereof may be utilized in treatment protocols.